The present invention relates to providing SPR sensors capable of assaying a plurality of samples simultaneously, methods for their production, measuring assemblies for scanning the sensors in accordance with the invention in parallel as well as to their use in the search for active agents and in high-throughput screening (HTS).
Continuing progress in automating the search for active agents has resulted in the question of miniaturization and parallelization gaining ever-increasing interest. Miniaturizing sample containers and the apparatus used in synthesis causes a plurality of substances to be assayed in ever-decreasing volume. This is why it is necessary in achieving novel detector and sensor systems to configure them so that several measurements can be run simultaneously in parallel, or a large number of samples assayed in sequence in shortest time whilst minimizing the volume of the substance needed therefor (high-throughput screening). Salient to this is enhancing the degree of automation.
There is furthermore a need to provide also the sensors used for analysis in a parallel and miniaturized format so that assaying a plurality of samples in shortest time and with minimum volume and consumption is achievable in thereby boosting the throughput of the substances to be characterized.
One highly sensitive measurement method for boundary layer characterization is known which is termed surface plasmon resonance (SPR) spectroscopy in pertinent literature. This method is based on optical excitation of surface plasmons along the boundary layer of films of metal.
It is usual in this arrangement to detect the light reflected by a thin gold film. When the condition for resonance is suitable (angle of incidence and wavelength of the light and film thickness of the gold film) the intensity of the reflected light decreases. In absorption of the light, excitation of the charge density waves of the electron gas occurs at the gold surface. These charge density waves are called plasma oscillations, their quantized excitation states plasmons.
To observe the resonance there are two methodic approaches. Either use is made of monochromatic light and the intensity of the reflected light is recorded as a function of the angle of incidence, or the angle of incidence is maintained constant and the wavelength of the light is varied. In both cases there is a shift in the location of resonance when the refractive index of the medium changes on the side of the gold film facing away from the incident light.
These methods are described in prior art as detailed, among others, by Striebel, Ch; Brecht, A; Gauglitz, G in Biosensors and Bioelectronics 9 (1994), 139-146. The resonance conditions for surface plasmon excitation greatly depend on the optical properties of the dielectric surrounding the metal film. Determining the refractive index or film thickness of thin dielectric films is basically possible with high accuracy by known prior art.
SPR spectroscopy is finding increasing application e.g. in biochemical analysis by making it possible to assay the interaction between partners (for example in the biomolecular reactions of antibodies and antigens) directly and without marking. For this purpose, an interaction partner (e.g. ligand) is immobilized on the surface of the metal, the other interaction partner (e.g. analyt) directed in solution over the surface. The interaction can be directly verified as the increase in film thickness via the change in the refractive index.
One task often involved in the miniaturization and parallel measurement of many samples is bringing the sensor sites into contact with fluid without, for example, cross-contamination occurring.
In the search for active agents conventional SPR sensors employ a prism coated with a thin film of metal. The sample to be assayed is brought into contact with the metal or the modified metal surface, and the SPR reflection spectrum of the sample is measured by coupling light into the prism and measuring the intensity of the reflected light as a function of the angle of incidence (cf. Chapter 8, xe2x80x9cSmall Molecule Drug Screening based on Surface Plasmon Resonancexe2x80x9d in Advances in Drug Discovery Techniques, John Wiley and Sons Ltd, London 1998).
A parallel approach to analyzing a sample array is SPR microscopy (SPM) (see e.g. EP 388 874 A2 or M. Zizlsperger, W. Knoll, Progr. Colloid Polym. Sci. 1998, 109, pp. 244-253) involving coating various sites on various samples of a gold surface applied to a prism and obtaining an image of the gold surface at the SPR angle on a CCD chip. During the measurement the angle is altered by a mechanical scanner. This method is, however, restricted to objects of small diameter.
A more recent SPR method is disclosed in WO 94/16312 A1 in which detecting the binding of small amounts of substance is achieved by optical fibers partially coated with a gold film. However, here too, the problem still exists in designing a sensor array required to assay a plurality of samples in parallel in accordance with this principle. Such an array of gold-coated fibers is, on the one hand, expensive and highly sensitive to mechanical stress, and, on the other, producing the array in parallel as proposed therein is difficult to achieve technically.
Optical fibers are also employed as it reads from WO 98/32002 A1. To protect them from being damaged physically the fiber cable is housed in a pipette. To achieve an array it is proposed to use a series arrangement of such pipettes. However, miniaturizing such an arrangement is difficult to achieve, especially for parallel measurement of many different samples.
WO 97/15819 A1 describes a dual-channel sensor comprising an objective mount as used in microscopy. Supplying fluid to the gold sensor site is achieved with the aid of a flow-through cuvette. Such a configuration is complicated especially in miniaturized HTS.
Known from U.S. Pat. No. 5,485,277 is a SPR sensor array using a waveguide configured so that multiple reflection occurs therein in thus simplifying signal analysis. Several spatial channels, e.g. a sensor channel and a reference channel, are employed in the measurement. There is no mention, however, of how the separate channels are generated.
Known from DE-196 15 366 A1 is a method and a means for simultaneously assaying a plurality of samples e.g. in a matrix arrangement. Separating the samples is done by applying the samples spatially separated.
WO 99/41594 describes a SPR system in which for a better time resolution the material properties of sites bordering a full-size SPR-sensitizing coating are modified to permit determining the intensity of the radiation reflected by the surface resolved in time or space.
WO 90/05295 describes a SPR system comprising a plurality of sensor surfaces. The SPR sensor system consists of a plate of glass coated with a film of metal, covered in turn by a dielectric film. The ligands making measurement possible are applied to the dielectric film. Separate sensor sites are generated by the separate application of the ligands.
A further possibility of simultaneously assaying a plurality of samples is described in WO 99/60382 A1 from which the preamble of the present claim 1 is known. In the arrangement as described several strip-type lightguides are arranged at a defined spacing on a planar backing and provided with a thin film of metal to permit excitation of such plasmons, whereby means are provided which separate the sensitized sites of the individual thin metal films by interruption thereof such that each lightguide can be assigned one sample only.
The lightguide described in WO 99/60382 A1 is fixed to a backing plate. This necessitates very high precision in production. The light-guiding film is deposited in a separate coating step. There is a risk of inhomogeneities materializing from one sensor to the other. Should solvent gain access to the interlayer between backing and light-guiding film problems may be encountered in the bonding of the deposited film.
The present invention is based on the objective of providing SPR sensors which are preferably planar, and measuring assemblies having a simpler configuration, as well as on methods for production thereof which are more cost-effective than those of known prior art in permitting simultaneous assaying of a plurality of samples. More particularly the invention is intended to create SPR sensors which include a light-guiding film containing at least two sensor surface areas as well as providing an array of at least two SPR sensors in avoiding the disadvantages of WO 99/60382 A1.
This objective is achieved by the characterizing features of claim 1 and by the subject matter of the parallel claims. Preferred aspects are described in the sub-claims.
In accordance with the invention there is now provided for an SPR sensor suitable for assaying a plurality of samples in parallel a self-supporting pattern in which a substrate backing SPR sensor sites is itself radiation-conductive and itself contributes towards beaming the radiation for SPR assaying.
Radiation in this sense is understood to be any radiation, especially electromagnetic radiation, in the range IR to visible suitable for excitation of plasmons. This radiation as preferred is sometimes also simply referred to as light without inferring any restriction to visible light, however.
The configuration of an SPR sensor in accordance with the invention overcomes the disadvantages of prior art (more particularly of WO 99/60382 A1) as regards the production problems since a plurality of radiation-conductive SPR sensor sites can now be provided in a single sensor without a separate backing needing to be provided. This not only simplifies production of the SPR sensor in accordance with the invention, but also results in SPR sensors which are more compact than known generic sensors. This facilitates, among other things, integrating a plurality of SPR sensors into a SPR sensor array.
As compared to prior art as set forth in DE-196 15 366 A1, U.S. Pat. No. 5,487,277, WO-99/41594 or WO-90/05295 the present invention features the difference and advantage that the SPR sensor sites are now separated by an interruption in the SPR-sensitzing film, resulting in a clear and explicit separation of the discrete sample sites right from the start with no need to use complicated ligand or sensing material coating techniques.
Making use of sensors or sensor arrays in accordance with the invention now makes it possible to assay as a function of the angle of incidence, which due to the small dimensions cannot be implemented with a lightguide in principle.
Although the SPR sensors in accordance with the invention can be assembled from initially separate SPR sensor sites and a body, the SPR sensors are preferably produced starting with a sole radiation-conductive substrate in which SPR sensor sites are formed which feature a SPR-compatible or SPR-sensitizing surface coating of suitable thickness (e.g. platinum, gold, silver, aluminum, copper, nickel or suitable alloys thereof) with the addition of an intermediate film promoting bonding (e.g. chromium) where necessary. These SPR sensor sites formed in the substrate into which radiation can be coupled and by which the radiation can be conducted for surface plasmon excitation in the SPR film and for communicating the modified radiation to an analyzer (i.e. modified by the SPR effect, in other words a reduction in intensity as a function of the wavelength or angle concerned as an indication of the dielectric properties of the outer side of the SPR film) are separated by separating means so that the radiation stemming from separated SPR sensor sites can be detected separately. These separating means also ensure that each SPR sensor site can be assigned a sample to be assayed, i.e. with no cross-contamination of neighboring SPR sensor sites.
In accordance with a first basic configuration the separating means are formed by depositing protuberances on the surface of the substrate. These are located between the SPR films, the substance from which the protuberances are made being selected so that no spreading of radiation occurs in the regions of the substrate between the SPR sensor sites, e.g. by such radiation being absorbed by this substance. For this purpose, the refractive index of the substance needs to be suitably selected to prevent a total reflection in the intermediate regions and the substance needs to have an absorbing or damping effect for the radiation. When the substrate is made of glass, e.g. silicon or a silicon compound is suitable for this purpose. In addition, the protuberances need to be selected so that they represent a fluid barrier so that no cross-contamination between neighboring samples or sensor sites occurs.
As evident, in this first basic configuration the substrate itself forms the backing for the SPR sensor sites because these and the separating areas are integral components of the substrate which remains unchanged in its volume. It is merely the surface that is changed in defining the corresponding sensor sites or separating areas in the interior of the substrate. Accordingly, all and any conduction or guidance of radiation takes place in both the sensor sites and in the backing or substrate.
In accordance with a second basic configuration the separating means are formed by cut-outs in the material of the substrate between the SPR sensor sites. These cut-outs can be formed by any suitable technique, e.g. by sawing, milling, etching etc. This results in the SPR sensor sites forming fingers protruding from the remaining original substrate which in turn forms the backing for the sensor sites. These fingers are, however, connected to the remaining substrate or backing radiation-conductively and the radiation coupled into the sensor sites also passes through the backing. The second basic configuration has the special advantage that the protruding fingers, representing SPR sensor sites, can be arranged in a suitable matrix and such SPR sensors on a suitable matrix can be composed into sensor arrays capable of cooperating with multiwell plates of corresponding format in thus creating particularly effective SPR sensor arrays for high-throughput screening.